ABSTRACT

ABSTRACT

In the present investigation, in vitro callus induction and plant regeneration were done in ashwagandha (Withania somnifera L.).
Leaf segments and internodal segments were cultured for callus induction on MS media supplemented with different concentrations and combinations of plant growth regulators and additives. Between the two types of explants, leaf segment explants found to be more effective for callus induction. The range of callus formation varied from 10%-70% and the highest frequency was 70% found from leaf segment explants when the MS medium was supplemented with 2.0mg/l 2,4-D. MS medium with 2,4-D was the best growth regulator formulation for callas formation from both types of explants.
MS medium was supplemented with different concentrations and combinations of BA with NAA and Kin with NAA for shoot regeneration from leaf segment and internodal segment explants derived calli. Kin with NAA was found more effective than BA. with NAA for maximum shoot regeneration from leaf segment derived calli. The range of shoot regeneration varied form 10%-50%. The highest 50% shoot regeneration was observed from leaf segment explants derived calli. MS medium supplemented with 4.0mg/l KIN + 0.5mg/l NAA was the best for maximum shoot regeneration from type of explants in ashwagandha. The highest number of shoots per callus was also found in this media formulation.




CONTENTS
LIST OF TABLES
LIST OF FIGURES
INDEX OF PLATES
ABSTRACT

CHAPTER I: INTRODUCTION
1.1. ASHWAGANDHA: A GENERAL ACCOUNT
1.2. HISTORY AND DISTRIBUTION
1.3. MORPHOLOGY AND TAXONOMY OF ASHWAGANDHA
1.4. IMPORTANCE
1.5. CULTIVARS
1.6. CULTIVATION METHODS.
1.7. CALLUS INDUCTION AND PLANT REGENERATION IN ASHWAGANDHA.
1.8. REVIEW OF LITERATURE
1.9. OBJECTIVES

CHAPTER II: MATERIALS AND METHODS
2.1. MATERIALS
2.1.1. Plant Materials
2.1.2. Chemicals
2.1.2.1. Basal nutrient media
2.1.2.2. Growth regulators
2.1.2.3. Sterilent solution
2.1.3. Other Materials


2.2. METHODS
2.2.1. Media used for Tissue Culture Techniques
2.2.2. Preparation of Stock Solutions for Culture Media
2.2.3. Preparation of 1 Liter Culture Medium
2.2.4. Culture Techniques

CHAPTER III: RESULTS
3.1. EFFECT OF DURATION OF TIME OF 0.1% MERCURIC
CHLORIDE TREATMENT ON SURFACE STERILIZATION
3.2. CALLUS INDUCTION
3.2.1. Callus Induction from Leaf Explants
3.2.1.1. Effects of different concentrations of 2, 4-D on callus induction from leaf explants.
3.2.1.2. Effects of different concentrations of NAA on callus induction from leaf explants.
3.2.1.3. Effects of different concentrations and combinations of NAA with BA on callus induction from leaf explants.

3.2.2. Callus Induction from Internodal Explants
3.2.2.1. Effects of different concentrations of 2, 4-D on callus induction from internodal explants.
3.2.2.1. Effects of different concentrations and combinations of NAA with BA on callus induction from internodal explants.

3.3. SHOOT REGENERATION FROM CALLUS
3.3.1. Shoot Regeneration from Leaf Explants Derived Calli.
3.3.1.1. Effects of different concentration and combinations of BA with NAA on shoot regeneration from leaf explants derived calli.
3.3.1.1. Effects of different concentrations and combinations of KIN with NAA on shoot regeneration from leaf explants derived calli.
3.3.2. Shoot Regeneration from Internodal Explants Derived Calli.
3.3.2.1. Effects of different concentrations and combinations of BA with NAA on shoot regeneration from internodal explantsderived calli
3.3.2.2. Effects of different concentrations and combinations of KIN with NAA on shoot regeneration from leaf explants derived calli.


CHAPTER IV: DISCUSSION

CHAPTER V: SUMMARY

CHAPTER VI: REFERENCES

APPENDIX




CHAPTER I

1. INTRODUCTION
1.1. ASHWAGANDHA: A GENERAL ACCOUNT
Ashwagandha (Withania somnifera (L.) Dunal) also known as Indian ginseng, Winter cherry, is a member of solanaceae. It is an erect perennial shrubby plant with ovate leaves, cluster of small flowers, ovoid berry-like red fruits and tuberous root. Ashwagandha in Sanskrit means “horses smell” probably originating from the odor of its root which resembles that of sweaty horse (Withania somnifera information from NPGS/GRIN". Retrieved on 2008-02-16). The species name somnifera means “sleep-making” in Latin but it has been used for sexual vitality. Some herbalists refer to ashwagandha as Indian ginseng, since it is used in ayurvedic medicine in a way similar to that ginseng is used in traditional Chinese medicine.
It is a widely used medicinal species useful in the treatment of inflammatory conditions, tuberculosis, rheumatism, as a tonic, or as an antitumour agent (Chopra et al. 1958, Suffness and Douros 1982). It grows in India, Pakistan, Sri Lanka and Bangladesh. It is commercially cultivated in Madhya Pradesh (state of India).
In Ayurveda, ashwagandha is considered a rasayana herb that works on a nonspecific basis to increase health and longevity. The roots and berries of the plant are used in herbal medicine. In Ayurveda, the fresh roots are sometimes boiled in milk, prior to drying, in order to leach out undesirable constituents. The berries are used as a substitute for rennet, to coagulate milk in cheese making.
The main constituents of aswagandha are alkaloids and steroidal lactones. Among the various alkaloids, withanine is the main constituent. The other alkaloids are somniferine, somnine, somniferinine, withananine, pseudo-withanine, tropine, pseudo-tropine etc.
Although many American and Japanese firms have filed for grant of patents on formulations containing extracts of ashwagandha, however, very limited research work been done for the improvement of this important medicinal plant. Plant tissue culture has been emerging as a powerful new technique for plant improvement. This technique is being used for micropropagation, distant hybridization, genetic transformation, induction of somaclonal variation and production of selected secondary metabolites in bioreactor. These techniques especially somaclonal variation and production of withananine in bioreactor may have potential role in ashwagandha improvement.
1.2. HISTORY AND DISTRIBUTION
Ashwagandha is used for 4000 years plus in India. It is a very important herb in ayurveda, the traditional Indian medicine. It is used for tumors, inflammation (including arthritis) and a wide range of infectious diseases. The shoots and seeds are also used as food and to thicken milk in India.
Traditional uses of ashwagandha among tribal peoples in Africa include fevers and inflammatory conditions. Ashwagandha is frequently a constituent of Ayurvedic formulas, including a relatively common one known as shilajit.
It is distributed throughout the drier region of India, especially in wasteland ascending to an altitude of 2000 m in the Himalaya. Distributional range of W. somnifera is given below:
1.3. MORPHOLOGY AND TAXONOMY OF ASHWAGANDHA
Morphology
Ashwagandha is a small, woody evergreen shrub that grows up to the height of 0.5 m to 1.5m. It can be found growing in Africa, the Mediterranean and India. As a result of this wide growing range, there are considerable morphological variations in terms of local species.
(i) Stem: They are solid, branched and usually erect. Stem and branches are covered with minute star shaped hairs.
(ii) Leaves: Simple, up to 10 cm long, ovate, petiolate and alternate.
(iii) Root: Roots are 20-30 cm long and 6-12 mm in diameter with few (2-3) lateral roots of slightly smaller size, straight, unbranched. Outer surface is buff to grayish-yellow with longitudinal wrinkles and in the centre soft, solid mass with scattered pores.
(iv) Flower: The flowers of ashwagandha plants are hermaphrodite, small, about 1cm long, greenish or lurid yellow in color; borne together in short axillary clusters. Each flower contains five stamens, two celled pistil, five sepals and five petals.
(v) Fruit: Ovoid, berry-like, orange-red in color, 6 mm diameter, smooth, enclosed in an inflated and membranous calyx.
Taxonomic position of ashwagandha
Ashwagandha (Withania somnifera) is a perennial shrubby plant belongs to the family solanaceae and genus Withania. Solanaceae family has 90 genus. Withania contains about species. There are over 20 other species of Withania that occur in the dry parts of India, North Africa, Middle East and the Mediterranean. These include Withania coagulens and Withania simonii, the roots of which are sometimes used interchangeably with those of Withania somnifera.
The accepted botanical name Withania somnifera was used by Linnaeus.
1.4. IMPORTANCE
The use of plants for the alleviation and cure of disease is older than man himself since the animals ate plants of certain kinds when they fell ill and indeed they still do. Medicinal plants thus formed the basis and foundation stone of medicine and treatment of disease at the very beginning of human civilization. Since that time thousands of plants having medicinal virtues of one kind or the other have been discovered.
World Health Organization (WHO) consultative group on medicinal plants has formulated a definition of medicinal plants in the following way: "A medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes or which are precursors for synthesis of useful drugs." (Sofowora, 1982). It has now been established that the plants which naturally synthesize and accumulate some secondary metabolites like alkaloids, sterols, terpenes, flavonoids, saponins, glycosides, cyanogenics, tannins, resins, lactones, quinones, volatile oils etc. and contain minerals and vitamins possess medicinal properties.
World health Organization (WHO) has compiled a list of 21,000 medicinal plants used in different parts of the world. Phytochemical and pharmacological studies of some of these plants have already resulted in the discovery and the development of many important drugs.
Ashwagandha is an important medicinal plant because of having different active and pharmacological effect. List of active constituents of ashwagandha is given below:
Anaferine (alkaloid), anahygrine (alkaloid), beta-sisterol, chlorogenic acid (in leaf only), cysteine (in fruit), cuscohygrine (alkaloid), iron, pseudotropine (alkaloid), scopoletin, somniferinine (alkaloid), somniferiene (alkaloid), tropanol (alkaloid), withaferin A (steroidal lactone), withanine (alkaloid), withananine (alkaloid) and withanolides A-Y (steroidal lactones). The active pharmacological components of Withania somnifera are steroidal lactones of the withanolide type. (Roy and Gupta, 1994). The principal withanolide in Indian Withania somnifera are withaferin A and withanolide D (Chakraborti et al., 1974). The main constituents of ashwagandha are alkaloids and steroidal lactones. Both leaves and roots of the plant are used as the drug and steroidal lactones occur in both parts (Chopra et al. 1958; Menseen and Stapel, 1973). Among the various alkaloids, withanine is the main constituent. The other alkaloids are somniferine, somnine, somniferinine, withananine, pseudowithanine, tropine, pseudo-tropine, 3-a-glyoxytropane, choline, cuscohygrine, isopelletierine, anaferine and anahydrine. Two acyl steryl glucoside viz. sitoindoside VII and sitoindoside VIII have been isolated from root. The leaves contain steroidal lactones, which are commonly called withanolides. The withanolides have C28 steroidal nucleus with C9 side chain, having six membered lactone ring.
Pharmacological Effects
Ashwagandha is reported to have anti-carcinogenic effects in animal and cell cultures by decreasing the expression of nuclear factor-kappaB, suppressing intercellular tumor necrosis factor and potentiating apoptotic signaling in cancerous cell lines.
Medicinal Uses
Historically, the plant has been used as an aphrodisiac, liver tonic, anti-inflammatory agent, astringent and more recently to treat bronchitis, asthma, ulcers, emaciation, insomnia and senile dementia. Clinical trials and animal research support the use of ashwaganda for anxiety, cognitive and neurological disorders, inflammation and Parkinson's disease. Ashwaganda's chemopreventive properties make it a potentially useful adjunct for patients undergoing radiation and chemotherapy. Ashwaganda is also used therapeutically as an adaptogen for patients with nervous exhaustion, insomnia and debility due to stress and as an immune stimulant in patients with low white blood cell counts.
In Ayurveda ashwaganda is considered a rasayana herb, which works on a nonspecific basis to increase health and longevity. This herb is also considered as an adaptogen which is a nontoxic herb that works on a nonspecific basis to normalize physiological function, working on the HPA axis and the neuroendocrine system. The roots and berries of the plant are used in herbal medicine. In ayurveda, the fresh roots are sometimes boiled in milk, prior to drying in order to leach out undesirable constituents. The berries are used as a substitute for rennet, to coagulate milk in cheese making.
Seven American and four Japanese firms have filed for grant of patents on formulations containing extracts of the herb ashwagandha. Fruits, leaves and seeds of the Indian medicinal plant Withania somnifera have been traditionally used for the Ayurvedic system as aphrodisiacs, diuretics and for treating memory loss. The Japanese patent applications are related to the use of the herb as skin ointment and for promoting reproductive fertility. The U.S based company Natreon has also obtained a patent for ashwagandha extract.
Another US establishment, the New England Deaconess Hospital, has taken a patent on an ashwagandha formulation claimed to alleviate symptoms associated with arthritis (Ashwagandha next on patent hunters list, Hindu Vivek Kendra archive of the Times of India May 16, 2001).
Other uses:
Ashwagandha is used as the substitute of the soaps. The leaves are insect repellent. Ashwaganda mixed with the almond oil and rose water is used as the facial toner. Ashwagandha is taken with the milk, sugar or honey as it promotes deep sleep.
1.5. CULTIVARS
A lot of intra-specific variability is reported in Withania somnifera growing in different parts of India. The characteristic genetic divergence is very much predominant within the distribution patterns of the genotypic and phenotypic variability. Five morphologically distinct ecotypes are reported. An improved strain WSR is developed and released for commercial cultivation by RRL, Jammu.
1.6. CULTIVATION METHODS:
(a) Land Preparation
Land is ploughed 3-4 times and planked twice to make the tilth fine and weed free. After leveling it is divided into beds of suitable size preferably 6m ´ 4m having proper irrigation channels.


(b) Seed sowing
The seed is sown from August to September after the heavy rains are over. Sowing is done by broadcast or line sowing method, in the field having sufficient moisture. Germination of seeds varies form 60-65%. Seed takes 10-12 days to germinate and germination is complete within a month. 2-kg seed is enough to cover one hectare area. When the seedlings are at 2-4 leaf stage, they should be thinned, maintaining a distance of 10 ´ 10 cm in the field.
(c) Irrigation
Ashwagandha is a rained crop and requires little irrigation after the crop get established. However, additional irrigation helps in better root growth.
(d) Fertilizer schedule
For this crop, application of chemical fertilizers is not recommended as its use invariably leads to a much branched and fibrous roots which are not good for commercial point of view.
(e) Pests and Diseases
Damping off is a major disease in Withania somnifera at seedling stage and results in heavy seedling mortality under field condition. However, it can be controlled by application of Dithane M-45 (0.3%) as foliar spray.
(f) Weed management
In the early stages of crop establishment, regular weeding is required, but once the crop picks up growth, the weeds are suppressed & weeding is done as and when required.
(g) Harvesting
The crop is harvested for roots in March-April, after six months of growth. After harvesting the roots are cleaned, cut into small pieces and graded according to diameter of the root. 1-2 cm diameter roots are the best category in the grading. 8-10 qtls of dry root/ha are obtained in case of WSR strain evolved by Regional Research Laboratory, Jammu.
1.7. CALLUS INDUCTION & PLANT REGENERATION IN ASHWAGANDHA
Plant cell and tissue culture techniques have become popular and being applied to solution of many agricultural and forestry problems (Rao and Lee, 1986). Callus tissue means an unorganized proliferative mass of cells produced from isolated plant cell, tissues or organs when grown aseptically on artificial nutrient medium in glass vials under controlled experimental conditions. Tissue and cell cultures are being explored as innovate breeding method for the genetic modification and improvement of plants. These techniques included in vitro propagation of desirable genotypes through anther culture, protoplast culture, protoplast fusion etc. Efficient plant regeneration methods are required for these techniques to be useful in crop improvement. Callus induction and development involve a complex relationship among the explants used to initiate the callus, the composition of the medium and the environmental conditions during the culture period. Induction of callus is one of the major steps for genetic transformation. When non-dividing quiescent cells from differentiated tissue are grown on a nutrient medium that supports their proliferation, the cells first undergo certain changes to active to meristematic state and forming un-differentiated callus tissue is termed dedifferentiation. A multicellular explant generally comprises cells of diverse types. As a result the callus derived from it would be heterogeneous with respect to the ability of its component cells to form a whole plant or plant organs which is called re-differentiation (Bhojwani and Razdan, 1983). Roja et al. (1991) reported callus formation from axillary meristem explants in MS medium with 2,4-D (2.0 mg 1-1) whilst Baburaj and Gunasekaran (1995) have also reported callus induction from leaf explants of W. somnifera using MS medium supplemented with NAA (2.0 mg 1-1) and KIN (0.5mg 1-1).
Shoot regeneration from different explants (leaves and internodes) can be divided into three phases: initiation of callus, initiation of shoots on the callus and shoot differentiation to whole plants (Hulme et al., 1992). Callus formation is promoted by maintenance of explants in the presence of both cytokinins and auxins.
1.8. OBJECTIVES
General objectives of this study are to establish a standard in vitro culture protocol for the induction of callus and successive plant regeneration from the target plant. The specific objectives of this study are as follows:
1. Standardization of surface sterilization for in vitro culture establishment.
2. Standardization of culture media formulations for callus culture.
3. Standardization of culture media formulations for plant regeneration through somatic embryogenesis and organogenesis.
4. Standardization of rooting media for plantlet production.
5. Field evaluation of the regenerated plant.

CHAPTER II

2. MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 PLANT MATERIALS
Seeds of Withania somnifera (L.) Dunal plants used in the present study were collected from the garden inside 3rd science building,, Rajshahi University.
2.1.2. CHEMICALS
The following chemical compounds were used in the present investigation:
2.1.2.1. BASAL NUTRIENT MEDIA
In this investigation MS (Murashige and Skoog, 1962) medium was used for the purpose of callus induction, shoot proliferation from callus and root induction.
2.1.2.2. GROWTH REGULATORS
Plant Growth Regulators (PGRs) are essential for good growth of cultured tissue and callus. The following growth regulators were used in the present investigation:
A. Auxins: Auxins promote cell enlargement and root initiation (Kyte, 1987).
i) a-napthalene acetic acid (NAA)
ii) 2, 4- Dichlorophenoxy acetic acid (2, 4-D)
B. Cytokinins: Cytokinins promote cell division and shoot induction (Kyte, 1987).
i) 6-benzyl adenine (BA)
ii) 6- furfuryl amino purine or kinetin (KIN)
2.1.2.3. STERILENT SOLUTION
HgCl2 Solution at 0.1% was used for surface sterilization of plant materials. To prepare 0.1% solution, 0.2 g of HgCl2 was dissolved in 200ml sterilized distilled water.
All chemical compounds used as macro and micronutrients in the present study were reagent grade (GPR) products of either Riedel-de-Haen, Germany, BDH, England or E-Mark Germany.
2.1.3 OTHER MATERIALS
Culture vessels such as test tube (150mm ´ 25mm), bottle (12mm ´ 5mm), conical flask (1250ml, 1000ml), measuring cylinders, glass rods, pipette, pipette pumps, parafilm, cotton plugs, forceps, marker pen, spirit lamp, scalpel, electronic balance, autoclave machine, pH meter, myoseasser, micro-wave oven, magnetic stirrer, laminar airflow machine etc. were also used in the present experiment.
2.2. METHODS
In tissue culture technique plants are regenerated inside test tubes, conical flax and in other glass vessels. Therefore, it is required to create a suitable environment (which may be termed as micro-environment) inside those glass vessels so that the plants propagated inside may have suitable support to stand erect and sufficient amount of O2 and CO2 for respiration and photosynthesis. It is some what specialized skillful job and requires adapting some special methods. Experimental methods carried out for this experiment were accomplished through following steps:
2.2.1. MEDIA USED FOR TISSUE CULTURE TECHNIQUES
In the present investigation, different culture media with various growth regulators and additives were used for various purposes were as follows:
2.2.1.1. MEDIA FOR CALLUS INDUCTION
The media used for callus induction were as follows:
i) MS (Murashige and Skoog, 1962) medium supplemented with different concentrations of 2, 4-D, NAA singly. 30 g/l sugar was added as carbon source and the medium was solidified with 6 g/l agar.
ii) MS (Murashige and Skoog, 1962) medium supplemented with different concentrations and combinations of NAA and BA.
2.2.1.2. MEDIA FOR PLANT REGENERATION
The media used for plant regeneration were as follows:
i) MS (Murashige and Skoog, 1962) media supplemented with different concentrations of BA and NAA having 30 g/l sucrose and 6 g/l agar.
ii) MS (Murashige and Skoog, 1962) media supplemented with different concentrations and combinations of KIN and NAA having 30 g/l sucrose and 6 g/l agar.
iii) MS (Murashige and Skoog, 1962) medium without hormone.

2.2.2. PREPARATION OF STOCK SOLUTIONS FOR CULTURE MEDIA
In the first step of the preparation of culture medium stock solutions were made. Various constituents of the respective nutrient medium were prepared into stock solutions for ready use during the preparation of media for different experiments. As different constituents were required in different concentrations, stock solutions of macro-nutrients, micro-nutrients, organic compounds (vitamin and amino acids) and growth regulators were prepared separately.
STOCK SOLUTION -I
NH4NO3 33g
KNO3 38g
KH2PO4 3.4g
The above listed nutrients were weighted accurately with the help of an electronic balance and dissolved in 200ml of distilled water. The final volume was made up to 400ml (for 20 liters). This stock solution was stored in a refrigerator at 4°C for several weeks.
STOCK SOLUTION-II
MgSO4. 7H2O 7.4g
This chemical was weighted accurately with the help of an electronic balance and dissolved in 100ml of distilled water. Then total volume was made up to 400ml (for 20 liters) by further addition of remaining 300ml of distilled water. Then poured into a bottle and stored in a refrigerator at 4° C for several weeks.
STOCK SOLUTION-III
CaCl2.2H2O 8.8g
This chemical was weighted accurately with the help of an electronic balance and dissolved in 100 ml of distilled water. Then total volume was made up to 400 ml (for 20 liters) by further addition of remaining 300ml of distilled water. Then poured into a bottle and stored in a refrigerator at 4° C for several weeks.
STOCK SOLUTION-IV
FeSO4.4H2O 0.556g
Na2-EDTA 0.746g
FeSO4.4H2O and Na2-EDTA were weighted accurately and dissolved separately in 175ml of distilled water by heating and constant stirring. Then the two solutions were mixed and pH was adjusted to 5.5. The final volume was made up to 400 ml (for 20 liters) by further addition of remaining distilled water. Then poured into a bottle and stored in a refrigerator at 4°C.
STOCK SOLUTION-V
MnSO4. 4H2O 0.446g
H3BO3 0.124g
ZnSO4. 7H2O 0.172g
The above listed nutrients were weighted accurately with the help of an electronic balance and dissolved in 200ml of distilled water. The final volume was made up to 400ml (for 20 liters). This stock solution was stored in a refrigerator at 4° C for several weeks.

STOCK SOLUTION-VI
KI 0.166g
CuSO4.5H2O 0.005g
NaMoO4. 2H2O 0.050g
CoCl2. 6H2O 0.050g
The above listed nutrients were weighted accurately with the help of an electronic balance and dissolved in 200ml of distilled water. Then the final volume made up to 400 ml and stored in a refrigerator at 4°C.
STOCK SOLUTION-VII
Myoinositol 5.000g
Nicotinic acid (vitamin B3) 0.025g
Pyridoxine-HCl (vitamin B6) 0.025g
Thiamine-HCl (vitamin B1) 0.025g
Glycine (amino acid) 0.100g
The above listed vitamins and amino acids were weighted accurately with the help of an electronic balance and dissolved in distilled water. The final volume was made up to 100ml. This stock solution was poured into a bottle and stored in a refrigerator at 0° C for several weeks.

STOCK SOLUTION OF PLANT GROWTH REGULATORS
In the nutrient media Plant Growth Regulators (PGRs) are essential for good growth of cultured tissue and organs. Different PGRs were used in the present investigation. Stock solutions of different plant Growth Regulators were prepared separately. Details of the preparation methods of stock solutions are shown in Table 2.2.1.
Table 2.2.1. Preparation of stock solutions for plant growth regulators
Growth regulators
Amount taken (mg)
Appropriate solvents
Final volume with distilled water (ml)
Strength of the stock solution
Auxin
NAA
2, 4-D
Cytokinin
BA
KIN


10
10

10
10

0.1N KOH (1ml)
70% ethanol (1ml)

0.1N KOH (1ml)
0.1N NaOH (1ml)

10
10

10
10

1
1

1
1
Plant Growth Regulators were dissolved in appropriate solvent as shown against each them (following the Sigma Plant Cell Culture Catalogue, 1992).
To prepare the stock solution of these growth regulators, 10mg of solid powdered growth regulators were placed into a clean test tube and then dissolved in specific solvent. The final volume of the solution was then made up 10 ml by adding distilled water. It was stored in a refrigerator in 4°-6° C temperatures for ready use in time.

2.2.3. PREPARATION OF 1 LITER CULTURE MEDIUM
Following steps were done for the preparation of 1 liter MS medium.
I. Assembling of the medium components
For the preparation of 1 litter MS medium, 20 ml of stock solution-1, 20 ml of stock solution-ll, 20 ml of stock solution-III, 20 ml of stock solution-IV, 20 ml of stock solution-V, 2 ml of stock solution-VI, 2 ml of stock solution-VII were added in 1 liter flask containing 500 ml distilled water and mixed well.
II. Addition of sucrose
30g of sucrose was added in the flask containing the stock solutions and was dissolved.
III. Addition of growth regulators
Stock solutions of growth regulators were added in appropriate concentrations and combinations in above solutions and were mixed well. Then the final volume of the mixture was made 1 liter by adding distilled water.
IV. pH of the medium
In all tests the pH of the medium was adjusted to 5.8 using a pH meter with the help of 0.1N KOH (where necessary) before addition of agar and sugar.
V. Addition of agar
To solidify the medium 6 g/l agar was added to the medium. Then the medium was heated for 8 minutes in a microwave oven (National, Japan) to melt agar completely.
VI. Medium dispensing to culture vessels
The prepared melted medium was disposed into culture vessels such as test tubes (130mm´ 25mm) or conical flasks (250ml), through separating funnel. The culture vessels were plugs, warped with cheesecloth which was inserted tightly at the mouth of culture vessels. The culture vessels were marked to designate specific hormonal supplements.
VII. Sterilization
Finally the culture vessels containing medium were autoclaved at 15lb/ inch2 pressure and at the temperature of 120°C- 121° C for 20 min to insure sterilization. Then the vessels with the medium were allowed to cool as vertically and then marked with a glass marker pen to indicate specific hormonal supplements and stored in the culture room for ready use.
2.2.4. CULTURE TECHNIQUES
The following culture techniques were employed in the present investigation:
2.2.4.1. CHOICE OF EXPLANTS
Plants of W. somnifera grown in Petri-dish were used as experimental material. For initiation of callus formation leaf segments and internodal segmants were used as explants.
2.2.4.2. CULTURE OF EXPLANTS FOR CALLUS INDUCTION
At first W. somnifera plant were surface sterilized with 0.1% HgCl2 for 2-4 min, washed 5 times with sterile double- distilled water and inoculated on agar-solidified MS medium supplemented with different concentrations of 2,4-D, NAA and BA, either alone or in combination. Cultures were maintained at (25±1)°C with a photoperiod with 40 (micro) mol m-2 s-1 provided by cool white fluorescent tubes. Callus was sub cultured after 25 days on the original callus-inducing medium (Rani and Grover, 1993).

2.2.4.3. SUBCULTURE OF CALLUS
After callus induction from the explants, the calli were transferred to the fresh callus inducing medium about 25 days interval for further proliferation and maintenance. The calli were subcultured in the same callus inducing medium after 22 to 29 days of induction and then the selected calli were transferred to regeneration media.
2.2.4.4. PLANT REGENERATION
Plants were regenerated by transferring the selected calli from leaf explants and internodal explants on MS, medium (semisolid medium) supplemented with two combinations of plant growth regulators KIN+ NAA and BA+ NAA for shoot regeneration. The cultures were incubated at 25±1°C under white light in dark conditions. After 3-4 weeks differentiation as well as shoot and root formation were observed. The number of calli producing shoots and total number of shoots were counted for each treatment. The shoots from the selected callus was excised and transferred to MS (without growth regulators) medium for further growth. The plantlets from each of individual callus were further multiplied by node culture using MS medium.
2.2.4.5. METHOD OF DATA RECORDING
Data were collected using the following parameters and the methods for data collection are given below:
a) For callus induction
I. Percentages of explants induced callus induced callus formation
Explants were cultured in 25 mm ´ 150 mm culture tubes containing media with different concentrations of growth regulators for callus induction. Some test tubes were contaminated which were separated and rejected. The rest of the test tubes in which explants remained fresh were maintained. After required days of culture; frequency of callus induction was calculated using the following formula:
Frequency of callus induction (%) =
II. Symbols used for callus induction
Cultured explants (leaf explants and internodal explants) which showed the induction of callus formation were counted after 6 weeks of culture. The colour and physical condition of callus varied in respect of explants type and growth regulator supplements. So different symbols were used to denote the different colour and degree of callus formation as given below:
Callus colour
Symbol
Brown
B
Light Brown
LB
Creamy
C
Greenish Brown
GrB
Light Green
LG
White
W
Yellow
Y
Light Yellow
LY

Degree of callus formation was marked in the following way:
Index
Description of callus formation
-
+
++
+++
No callus
Slight callus
Moderate callus
Massive callus

b) For shoot induction
I. Percentage of calli induced shoots
As mentioned earlier data on different parameters from different treatments of shoot proliferation were recorded after required days of culture. The percentages of calli induced shoots were calculated using following formula:
Shoots induced (%) =
II. Number of shoots per callus
Number of shoots per callus (explants) was computed after required days of culture.
Mean number of shoots per callus (explants) was calculated using following formula:
= Average number of shoots
= Summation
Xi = Total number of shoots
N = Number of observation
III. Length of the longest shoot
Length of the longest shoot was measured in cm scales for each explants. Average length of the longest shoot was calculated by using the following formula:
Length of the longest shoot =

CHAPTER III
3. RESULTS
The present investigation was carried out for callus culture and indirect regeneration of ashwagandha (Withania somnifera L.). The objectives of this investigation were to establish a protocol for callus induction and plant regeneration from different parts of ashwagandha plant. The results obtained from each of the experiments are described below.
3.1. EFFECT OF DURATION OF TIME OF 0.1% MERCURIC CHLORIDE TREATMENT ON SURFACE STERILIZATION
For the primary establishment of culture, surface sterilization of explants was essential because of presence of loose contaminants attached to the surface of the explants. Immature leaves of plants were used as primary explants after sterilizing their surface. Mercuric chloride (HgCl2) solution was used as surface sterilant but non-judicious application of HgCl2 solution for uncertain duration may lead to contamination or tissue killing.
So an experiment was undertaken for standardizing accurate duration of time for surface sterilization. For this purpose 0.1% HgCl2 was tested for different duration of time. Results obtained are shown in Table 3.1.
After 12 days of sterilization and incubations it was observed that the highest 95% of explants showed contamination free condition when they were treated for three minutes. But partial tissue killing was 5 %. So the survival rate was 90% (when they were treated for three minutes).
Treatments for 2-2.5 minutes were also proved to be efficient for the elimination of loose contaminants to some degree. However, when 0.1% HgCl2 was used for short duration (less than 2 minutes), the treatment failed to kill the micro organisms attached to the surface of explants.
Hundred percent contamination free tubes were observed when they treated for 3.5 to 4 minutes but tissue killing in these treatment ranged from 45%-80% and among survival rate ranged from 55% - 20%.
Table 3.1. Effect of HgCl2 treatment duration for surface sterilization of shoots of ashwagandha.
Treatment duration of 0.1% HgCl2 in min.
Total number of explants
Contamination rate after days
Tissue Killing
Number of contamination free explants
% of contamination free explants
% of survival
2
4
6
8
10
12
Complete
Partial
No
2
20
1
5
8
10
12
13
-
-
-
7
35
35
2.5
20
-
-
2
3
5
-
-
-
-
15
75
75
3
20
-
-
-
-
-
-
-
1
19
19
95
90
3.5
20
-
-
-
-
-
-
4
5
11
20
100
55
4
20
-
-
-
-
-
-
10
6
4
20
100
20




Time in minutes
Graphical representation of % of contamination free explants against time. 3.2. CALLUS INDUCTION
This investigation was carried out for the induction of callus from different explants (leaf and internodel) and their maintenance for further growth. Shoots were collected from Petri-dish grown plants and surface sterilized with 0.1% HgCl2 solution. leaves were cut into small pieces (3mm × 3 mm) from sterilized shoots and internodes were cut into same size (collected from in vitro grown plants) and used as explants in this investigation. Explants were cultured on MS medium solidified with agar and supplemented with different concentrations of 2, 4-D, NAA alone and in combinations of NAA with BA in order to find out the most suitable culture media formulation to induce maximum callus formation from the cultured explants. The rates of callus induction showed a great variation with different concentrations and combinations of plant growth regulators. Usually callus proliferation started from the cut surface of the explants and gradually covered the whole explant.
The growth of a cultured callus over a period of time is characterized by an increase in cell number, an increase in volume or mass and changes in biochemistry and cellular complexity. Percentage of explants induced callus, callus colour and degree of callus development - these three parameters were noted. The results of present piece of work are described below under the following separate heads.
3.2.1. CALLUS INDUCTION FROM LEAF EXPLANTS
The leaf explants of ashwagandha were cultured on MS medium supplemented with six different concentrations of 2, 4-D, NAA and six different combinations of NAA with BA. Results concerning with effects of these treatments on percentage of callus induction, degree of callus formation and callus colour are described bellow.
3.2.1.1. EFFECTS OF DIFFERENT CONCENTRATIONS OF 2, 4-D ON CALLUS INDUCTION FROM LEAF EXPLANTS
The leaf explants of ashwagandha were cultured on MS medium supplemented with six different concentrations of 2, 4-D ranging from 1.0 mg/l to 3.5 mg/l. The data were recorded after six weeks of culture and are presented in Table 3.2. The callus showed a great variation in response with treatment concentrations.
The highest 60% leaf explants were found to induce callus formation in media having 2.0 mg/l 2, 4-D. The calli in this media formulation were brown in colour and the degree of callus formation was massive. The second highest 50% leaf explants were induced to callus formation in media having 2.5 mg/l 2, 4-D. The calli in this treatment were brown in colour and the degree of callus formation was moderate. The lowest 10% of the explants induced to develop callus formation in media having 1.0 mg/l 2, 4-D. In this treatment the calli were white in colour and the degree of callus formation was very low.
Table 3.2. Effects of different concentrations of 2, 4-D in MS medium on callus induction from leaf explants of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after six weeks of culture.
Growth regulator (mg/l) 2, 4-D
% of explants induced callus
Callus color
Degree of callus formation
1.0
1.5
2.0
2.5
3.0
3.5
10
40
60
50
40
20
W
LB
B
B
B
B
+
+
+++
++
+
+

3.2.1.2 EFFECTS OF DIFFERENT CONCENTRATIONS OF NAA ON CALLUS INDUCTION FROM LEAF EXPLANTS
The leaf explants of ashwagandha were cultured on MS medium supplemented with six different concentrations of NAA ranging from 1.0 mg/l to 3.5 mg/l. The data were recorded after six weeks is presented in table 3.3. The response of callus greatly varied with treatment concentrations.
Among all the treatments of NAA, the highest 40% leaf explants induced callus when they were cultured on MS media supplemented with 2.5 mg/l NAA. Callus developed in this media formulation was yellow in colour and the degree of callus formation was moderate. The second highest 30% explants formed callus in media having3.0 mg/l NAA. Callus developed in this media formulation was light yellow in colour and the degree of callus formation was low. The lowest 10% explants induced callus in media having 1.5 mg/l NAA and the degree of callus formation was low. In this media formulation the colour of callus was white. In media having 1.0 mg/l NAA the explants did not show any response to induce callus.
Table 3.3. Effects of different concentrations of NAA in MS medium on callus induction from leaf explants of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after six weeks of culture.
Growth regulator (mg/l) NAA
% of explants induced callus
Callus color
Degree of callus formation
1.0
1.5
2.0
2.5
3.0
3.5

-
10
20
40
30
20

-
W
LY
W
LY
W
-
+
+
++
+
+

3.2.1.3. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBINATIONS OF NAA WITH BA ON CALLUS INDUCTION FROM LEAF EXPLANTS
The leaf explants of ashwagandha were cultured on MS medium supplemented with six different concentrations and combinations of NAA with BA. The response of callus greatly varied with culture media formulations. The results obtained are presented in Table 3.3.
Among all the treatment concentrations and combinations of NAA with BA, maximum 60% leaf explants induced to form callus in media containing 1.0 mg/l NAA +1.0 mg/l BA. The callus developed in this media formulation was greenish brown in colour and the degree of callus formation was massive. The second highest 50% explants induced to develop callus in media having 1.5 mg/l NAA+0.5 mg/l BA. The colour of callus developed in this media formulation was light brown and the degree of callus formation was moderate. The lowest 10% leaf explants induced to callus formation in media containing 0.5 mg/l NAA+0.5 mg/l BA. The colour of callus in this media formulation was white and the degree of callus formation was low.
Table 3.4. Effects of different concentrations and combinations of NAA with BA in MS medium on callus induction from leaf segment explants of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after six weeks of culture.
Growth regulators (mg/l) NAA + BA
% of explants induced callus
Callus color
Degree of callus formation
0.5 + 0.5
0.5 + 1.0
1.0 + 0.5
1.0 + 1.0
1.5 + 0.5
1.5 + 1.0
10
20
40
60
50
20
W
LG
LB
GrB
LB
LB
+
++
++
+++
++
++

3.2.2. CALLUS INDUCTION FROM INTERNODAL EXPLANTS
The internodal explants of ashwanandha were cultured on MS medium supplemented with different concentrations of 2, 4-D, NAA alone and in combinations of NAA with BA to observe the response of explants for callus induction. The response of callus greatly varied with treatment concentrations and combinations. The results are described below.
3.2.2.1. EFFECTS OF DIFFERENT CONCENTRATIONS OF 2, 4-D ON CALLUS INDUCTION FROM INTERNODAL EXPLANTS
Six different concentrations of 2, 4-D ranging form 1.0 mg/l to 3.5 mg/l were used in this record. After 6 weeks of culture the percentage of induced callus, callus colour and degree of callus formation were observed and recorded that are presented in Table 3.4.
Among all of the treatments, the highest 50% internodes were induced to form callus when they were cultured on 2.5 mg/l 2, 4-D containing MS medium. Callus developed in this media fromulation was brown in colour. The massive degree of callus formation was also recorded in the same media. The second highest 40% explants formed callus on MS media containing 3.0 mg/l 2, 4-D. The callus developed in this medium was also brown in colour and the degree of callus formation was moderate. The lowest callus formation observed in media having 1.5 mg/l 2, 4-D. In this case 10% explants induced callus. The colour of callus in this media was creamy and the degree of callus formation was low. The media supplemented with 1.0 mg/l 2, 4-D failed to induce the explants to form callus.
Table 3.5. Effects of different concentrations of 2, 4-D in MS medium on callus induction from internodal explants of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after six weeks of culture.
Growth regulator (mg/l) 2, 4-D
% of explants induced callus
Callus color
Degree of callus formation
1.0
1.5
2.0
2.5
3.0
3.5
-
10
30
50
40
20
-
C
LB
B
B
C
-
+
++
+++
++
+

3.2.2.2. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBANATIONS OF NAA WITH BA ON CALLUS INDUCTION FROM INTERNODAL EXPLANTS
The internodal explants of ashwagandha were cultured on MS medium supplemented with different concentrations and combinations of NAA with BA. Six different concentrations of NAA with BA were tested and these were 0.5 mg/l NAA+0.5 mg/l BA, 0.5 mg/l NAA+1.0 mg/l BA, 1.0 mg/l NAA+0.5 mg/l BA, 1.0 mg/l NAA +1.0 mg/l BA, 1.5 mg/l NAA+0.5 mg/l BA and 1.5 mg/l NAA+1.0 mg/l BA. The response of callus greatly varied with treatments which have been presented in Table 3.6.
Among all the concentrations and combinations of NAA with BA, maximum 50% explants induced callus in media having 1.0 mg/l NAA+1.0 mg/l BA. The callus developed in this media formulation was brown in colour and the degree of callus formation was massive. The lowest 10% explants induced callus formation in media containing 1.5 mg/l NAA+1.0 mg/l BA. The callus developed in this media formulation was brown in colour and the degree of callus formation was relatively low. When the explants were cultured in media having 0.5 mg/l NAA+ 0.5 mg/l BA, the explants did not show any response.
Table 3.6. Effects of different concentrations of NAA in MS medium on callus induction from internodal explants of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after six weeks of culture.
Growth regulators (mg/l) NAA + BA
% of explants induced callus
Callus color
Degree of callus formation
0.5 + 0.5
0.5 + 1.0
1.0 + 0.5
1.0 + 1.0
1.5 + 0.5
1.5 + 1.0
-
20
40
50
30
10

-
LB
B
B
B
B
-
+
++
+++
+
+



3.3. SHOOT REGENERATION FROM CALLUS
Different experiments were conducted to investigate plant regeneration ability through callus culture of ashwagandha. The calli derived from leaf explants and internodal explaants were subcultured in MS media supplemented with different concentrations and combinations of auxins and cytokinins for regeneration of plantlets. The results obtained are described according to types of explants under following separate heads.
3.3.1. SHOOT REGENERATION FROM LEAF EXPLANTS DERIVED CALLI
Calli derived from leaf explants were subcultured in MS medium supplemented with different concentrations and combinations of BA with NAA and KIN with NAA. The effects of these growth regulators on shoot regeneration, number of shoots per callus and length of the longest shoot were recorded after four weeks and the results are described below.
3.3.1.1. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBINATIONS OF BA WITH NAA ON SHOOT REGENERATION FROM LEAF EXPLANTS DERIVED CALLI.
The calli derived from leaf explants were subcultured in MS medium supplemented with six different concentrations and combinations of BA with NAA in order to find out the most suitable culture media formulation for shoot regeneration. A great variation was observed due to the difference of growth regulators. The results obtained after four weeks of culture are presented in Table 3.7.
Among all the formulations, the highest 40% calli induced to develop shoot in media having 3.0 mg/l BA + 0.1 mg/l NAA. The lowest 10% calli were recorded to induce shoot regeneration in media having 2.0 mg/l BA + 1.0 mg/l NAA and 3.0 mg/l BA + 1.5 mg/l NAA. The highest number of shoots per callus was recorded 7.1 when they were cultured in media having 3.0 mg/l BA + 0.1 mg/l NAA. The lowest number of shoots per callus was recorded as 3.2 when the media were supplemented with 2.0 mg/l BA + 1.0 mg/l NAA. The highest length of the longest shoot was recorded as 2.8 cm after four weeks of culture in media having 2.0 mg/l BA + 1.0 mg/l NAA. The lowest length of the longest shoot was recorded as 1.2 cm after four weeks of culture in media having 3.0 mg/l BA + 1.0 mg/l NAA.

Table 3.7. Effects of different concentrations and combinations of BA with NAA in MS medium on shoot regeneration from leaf explants derived calli of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after four weeks of culture.
Growth regulators (mg/l)
BA + NAA
% of calli regenerated shoots
No. of shoots/callus
Length of the longest shoot after 4 weeks (cm)
2.0 + 1.0
2.0 + 1.5
3.0 +0.1
3.0 + 0.5
3.0 + 1.0
3.0 + 1.5
10
20
40
30
20
10

3.2
4.1
7.1
6.2
5.3
4.6
2.8
2.2
2.3
1.5
1.2
1.5

3.3.1.2. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBINATIONS OF KIN WITH NAA ON SHOOT REGENERATION FROM LEAF EXPLANTS DERIVED CALLI.
The calli derived from leaf segments were subcultured in MS semisolid medium supplemented with six different concentrations and combinations of KIN with NAA in order to find out the most suitable culture media formulation for shoot regeneration. The effects of different concentrations and combinations of growth regulators on shoot regeneration were observed after four weeks of inoculation and the results are given in Table 3.8. The results obtained from the experiments are described below.
Among all the formulations of KIN with NAA, the highest 50% calli were induced to shoot regeneration in media having 4.0 mg/l KIN+0.5 mg/l NAA. The lowest 10% calli were observed to induce shoot regeneration in media having 3.0 mg/l KIN+0.1mg/l NAA. The highest number of shoots per callus was recorded as 5.9 in media having 4.0 mg/l KIN+0.5 mg/l NAA. The lowest number of shoots per callus was recorded as 2.1 in media having 3.0 mg/l KIN+0.1 mg/l NAA. The highest length of the longest shoot after four weeks of culture was recorded as 5.2 cm in media having 4.0 mg/l KIN+1.5 mg/l NAA. The lowest length of the longest shoot was recorded as 2.3 cm after four weeks of culture in media having 4.0 mg/l KIN+0.5 mg/l NAA.
Table 3.8. Effects of different concentrations and combinations of KIN with NAA in MS medium on shoot regeneration from leaf explants derived calli of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after four weeks of culture.
Growth regulators (mg/l)
KIN + NAA
% of calli regenerated shoots
No. of shoots/callus
Length of the longest shoot after 4 weeks (cm)
3.0 + 0.1
3.0 + 0.5
3.0 +1.0
4.0 + 0.5
4.0 + 1.0
4.0 + 1.5
10
20
30
50
30
40

2.1
2.6
3.2
5.9
3.1
4.3
3.9
3.3
4.2
2.3
2.7
5.2

3.3.2. SHOOT REGENERATION FROM INTERNODAL EXPLANTS DERIVED CALLI
Calli derived from internodal explants were subcultured in MS medium supplemented with different concentrations and combinations of BA with NAA and KIN with NAA. The effects of these growth regulators on shoot regeneration, number of shoots per callus and length of the longest shoot were recorded after four weeks and the results are described below.
3.3.2.1. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBINATIONS OF BA WITH NAA ON SHOOT REGENERATION FROM INTERNODAL EXPLANTS DERIVED CALLI
Calli derived from internodal explants were subcultured on MS semisolid medium supplemented with six different concentrations and combinations of BA with NAA in order to find out the most suitable culture media formulation for shoot regeneration and for suitable growth of shoots to get maximum number of shoots per callus. A great variation was observed due to different concentrations and combinations of growth regulators and the results obtained from this experiment are presented in Table 3.9.
Among all the formulations of BA with NAA, the maximum 30% calli induced to develop shoot regeneration in media having 3.0 mg/l BA + 1.0 mg/l NAA. The lowest 10% calli were recorded to show shoot regeneration in media having 2.0 mg/l BA + 1.0 mg/l NAA. The highest number of shoots per callus was recorded 4.6 in media having 3.0 mg/l BA + 1.0 mg/l NAA. The lowest number of shoots per callus was 2.1 in media having 2.0 mg/l BA + 1.0 mg/l NAA. The highest length of the longest shoot was recorded as 2.2 cm after four weeks of culture when the medium was supplemented with 2.0 mg/l BA + 1.5 mg/l NAA. The lowest length of the longest shoot was recorded as 1.3 cm after four weeks of culture in media having 3.0 mg/l BA + 0.1 mg/l NAA.
Table 3.9. Effects of different concentrations and combinations of BA with NAA in MS medium on shoot regeneration from internodal explants derived calli of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after four weeks of culture.
Growth regulators (mg/l)
BA + NAA
% of calli regenerated shoots
No. of shoots/callus
Length of the longest shoot after 4 weeks (cm)
2.0 + 1.0
2.0 + 1.5
3.0 +0.1
3.0 + 0.5
3.0 + 1.0
3.0 + 1.5
10
20
20
20
30
20
2.1
2.7
2.4
2.5
4.6
2.2
2.0
2.2
1.3
1.6
2.1
1.7

3.3.2.2. EFFECTS OF DIFFERENT CONCENTRATIONS AND COMBINATIONS OF KIN WITH NAA ON SHOOT REGENERATION FROM INTERNODAL EXPLANTS DERIVED CALLI
Calli derived from internodal segment explants of ashwagandha were subcultured on MS semisolid medium supplemented with different concentrations and combinations of KIN with NAA. The results obtained are presented in Table 3.10.
Among all the formulations, the highest 30% calli induced to develop shoot regeneration in media having 4.0 mg/l KIN + 0.5 mg/l NAA. The lowest 10% calli were recorded to develop shoot regeneration in media having 3.0 mg/l KIN + 1.0 mg/l NAA. The highest number of shoots per callus was recorded as 4.6 when the media were supplemented with 4.0 mg/l KIN + 0.5 mg/l NAA. The lowest number of shoots per callus was 2.1 when the media were supplemented with 3.0 mg/l KIN + 0.1 mg/l NAA. The highest length of the longest shoot after four weeks of culture was recorded as 4.5 cm in media having 3.0 mg/l KIN + 1.0 mg/l NAA. The lowest length of the longest shoot after four weeks was 2.4 cm when the media were supplemented with 3.0 mg/l KIN + 0.1 mg/l NAA.
Table 3.10. Effects of different concentrations and combinations of KIN with NAA in MS medium on shoot regeneration from internodal explants derived calli of ashwagandha. In each treatment 10 explants were inoculated. Data were recorded after four weeks of culture.
Growth regulators (mg/l)
KIN + NAA
% of calli regenerated shoots
No. of shoots/callus
Length of the longest shoot after 4 weeks (cm)
3.0 + 0.1
3.0 + 0.5
3.0 + 1.0
4.0 + 0.5
4.0 + 1.0
4.0 + 1.5
10
20
20
30
20
20
2.1
2.3
2.2
4.6
3.9
3.5
2.4
2.6
4.5
4.0
3.7
4.2


CHAPTER IV
DISCUSSION
In recent years, propagation of numerous plants by tissue culture has become accepted in commercial practice. As conventional breeding has potential limitation (Chaudhury, 1994), the techniques of plant cell and tissue culture have become more popular and useful methods in many temperate countries, which are being applied to solution of problems in many agricultural and forestry (Rao and Lee, 1986).
The difference between tissue culture and conventional breeding involve the use of smaller propagules, the provision of an aseptic and artificial environment and substantially faster plant multiplication. Currently, cloning by in vitro techniques is especially valuable for propagation of many ornamental, medicinal, vegetable and fruit crops.
Ashwagandha (Withania somnifera (L.) Dunal.) is a widely used medicinal species useful in the treatment of inflammatory conditions, rheumatism, as a tonic, or as an anti-tumour agent (Chopra et al.1958, Suffness and Douros, 1982). Propagation is mainly by, but seed viability is limited to one year Due to poor viability of stored seed and a lack of a protocol for in vitro (mass) multiplication, the present study was undertaken to examine the potential of different explants to respond under in vitro conditions with the possibility of developing a protocol for the in vitro multiplication of Withania somnifera.
In the present investigation callus induction and plant regeneration wre done at Plant Breeding and Gene Engineering Laboratory of Rajshahi university in Bangladesh using different parts (leaf segments and internodal segments) of ashwagandha. The results obtained in the present investigation are discussed in the following paragraph with an endeavour to justify them.
4.1. CALLUS INDUCTION
The term callus refers to tissue arising from the disorganized proliferation of cells from segments (explants) of plant organs. Callus formed during in vitro culture has some similarities to tissue arising in vivo injury to plants (so called wound callus). However, there often are differences in morphology, cellular structure, growth and metabolism between callus derived through tissue culture and natural wound callus.
To initiate callus from different explants, an exogenous supply of growth regulators are often required. Exogenous supplies of auxin and often in combination with cytokinin to medium are essential for callus induction. Rao and Lee (1968) reported that intermediate levels of auxin and cytokinin usually promote callusing. But many other factors like genotypes, compositions of the nutrient medium, physical growth factors such as light, temperature, humidity etc. are important for callus induction (Pierik, 1987).
For callus culture of dicot plants there are many reports with many species. On the contrary, for monocot plants, it has been believed that callus induction is very difficult, because they have no secondary growth which occurs through the activity of vascular combium (Maeda, 1980). Auxins are usually required for the induction of callus from a variety of tissue explants, except cambial tissues that can proliferate without an exogenous supply of auxin (Minocha, 1987). It has now been well established that any tissue can be changed into callus if it is cultured on a suitable defined medium under controlled conditions.
In the present experiment explants were collected from Petri-dish grown plant of ashwagandha. Two types of explants (internodal segments and leaf segments) were used. Explants were cultured on MS medium supplemented with different concentrations of 2, 4-D, NAA and in combinations of NAA with BA in order to find out the most suitable culture media formulation to induce the explants to develop maximum callus. Among the different auxins (2,4-D, NAA) and cytokinin (BA) were tried singly or in combination in various concentrations, 2, 4-D was found to be best for callus induction from both types of explants.
Among all the treatments of 2, 4-D, maximum 60% callusing rate was found in media having 2 mg/l 2, 4-D from leaf segment explants. The rate of callus induction from internodal segment explants was lower than leaf base segment explants. Maximum 50% callusing rate was found from internodal segment explants in media having 2.5 mg/l 2, 4-D. The degree of callus formation from internodal segment explants was lower than leaf segment explants.
Different concentrations of NAA were used to induce callus formation from leaf segment explants. As the response was not sutisfactory, so further it was not used to induce callus from internodal segment explants. Among all the treatments of NAA, the highest 40% explants induced to callus formation in media having 2.5 mg/l NAA. The degree of callus formation was not satisfactory.
According to the experimental results, different concentrations, and combinations of auxin and cytokinin (BA + NAA) were also found to induce callus from leaf segment explants and internodal segment explants of ashwagandha.
The highest 50% callusing rate was recorded from leaf segment explants in media 1.0 mg/l NAA+ 1.0 mg/l BA. The degree of callus formation was satisfaction.
The callusing rate from internodal segment explants was highest 50% in media having 1.0 mg/l NAA + 1.0 mg/l BA. The degree of callus formation was not satisfactory.
Therefore in the present investigation, it was clearly focused that MS medium containing 2.0mg/l 2,4-D was the best medium for callus induction from leaf segment explants and MS medium containing 2.5 mg/l 2,4-D was the best medium for calllus induction from internodal segment explants in ashwagandha.
Many workers obseerved 2, 4-D as the best auxun for callus induction as common as in monocot and even in dicot (Evans et al., 1981; Lu et al., 1982; Ho and Vasil, 1983; Jaiswal and Narayan, 1985; Chee, 1990). Oka and Ohyma (1976), also obtained good amount of callus in media with 2, 4-D than other auxins in case of a dicot plant mulberry. Raja et al. (1991) reported that MS medium with 2, 4-D (2mg/l) is the best medium to induce callus from axillary meristem explants in ashwagandha.
4.2. PLANT REGENERATION
Calli derived from leaf explants and internod explants of ashwagandha were subcultured for shoot regeneration in MS medium supplemented with different plant growth regulators such as BA with NAA and KIN with NAA in different concentrations and combinations. After subculture of callus, shoot regeneration started within 21-28 days of culture along with more callus proliferation.
The necessity of cytokinin for shoot initiation is well established (Torrey, 1968, Englke et al. 1973, Narayanswamy 1977, Back and Coponetti, 1983 and Evans et al. 1984). But shoot regeneration from nodal, internodal and leaf explants derived callus do not seem to be an easy process. Regeneration rate of plantlet from callus in ashwagandha is generally poor.
The media with different concentrations and combinations of BA with NAA and KIN with NAA were found effective for shoot regeneration of ashwagandha from leaf explants and internodal explants derived callus. Two successive culture phases are required for inducing multiple shoots as reported by many earlier researchers (Conover and Litz 1978).
Between the two combinations, KIN+NAA was proved to be more effective than BA+NAA for maximum shoot induction from leaf explants derived callus of ashwagandha. Among the different concentrations and combinations of BA with NAA, the media having 3.0 mg/l BA +1.0 mg/l NAA were found optimum 40% for leaf explants responded to develop shoots when cultured in this media composition. The maximum number of shoots per callus was also recorded in this media formulation. The highest length of the longest shoot was 2.8 cm in media having 2.0 mg/l BA + 1.0 mg/l NAA.
Among all the concentrations and combinations of BA with NAA, the media having 3.0 mg/l BA + 1.0 mg/l NAA was found optimum 30% shoot regeneration from internodal explants derived calli. The maximum number of shoots per callus was also recorded in this media composition. The highest length of the longest shoot was 2.2 cm having 2.0 mg/l BA + 1.5 mg/l NAA.
MS medium was supplemented with different concentrations and combinations of KIN with NAA to induce shoot regeneration from the internodal and leaf explants derived calli responded to shoot regeneration in media fortified with 4.0 mg/l KIN +0.5 mg/l NAA. The maximum number of shoots per callus was also found in this media composition. The highest length of the longest shoot was 5.2 cm in media having 4.0 mg/l KIN + 1.5 mg/l NAA.
The regeneration rate of internodal explants derived calli was lower than that of leaf explants derived calli. The highest 30% internodal segment explants derived calli induced to regenerate shoots in media having 4.0 mg/l KIN + 0.5 mg/l NAA. The maximum number of shoots per callus was also found in the same media formulation. The highest length of the longest shoot from internodal segment derived callus was 4.5cm in media having 3.0 mg/l KIN + 1.0 mg/l NAA.
In the present investigation, the media fortified with 3.0 mg/l BA +1.0 mg/l NAA was found optimum from internodal explants derived calli for maximum shoot regeneration and highest number of shoots per callus. Later it was noted that KIN with NAA showed better performance than BA with NAA for maximum shoot regeneration and shighest number of shoots per callus on leaf segment explants derived calli. A better result was found from leaf segment explants derived calli in media having 4.0 mg/l KIN +0.5 mg/l NAA. In the present investigation MS medium supplemented with 4.0 mg/l KIN +0.5 mg/l NAA was recorded as optimum concentration and combination for maximum shoot regeneration and highest number of shoots per callus from both type of explants. Baburaj and Gunasekaran (1995) used KIN with NAA in MS medium for shoot regeneration from callus in Withania somnifera. Sultana (2001) used KIN with NAA in MS medium for shoot regeneration from callus in Solanum tuberosum. Suh and Park (1986) recorded that KIN with NAA in MS medium also stimulated proliferation and elongation of shoots in garlic (Allium sativa L.). Ratio of cytokinin and auxin seems to play an important role in the morphogenic differentiation of cultured explants, as suggested by Murashige and Skoog (1962), Steward et al. (1969), Thomas and Street (1970), Pareek and Chandra (1981), Beck and Coponetti (1983) and Haider (1992). This differential response with regard to morphogenic response of Solanum explants may be due to the genotype differences of the plant material used in the investigation.
Therefore, from the following discussion it was establiushed that an in vitro response in relation to callus induction and shoot regeneration is the result of a complex interaction involving the physiological status of the explants, the genotype and the composition and concentrations of the components of the culture media.
CHAPTER V

SUMMARY
The present investigation was undertaken with a view to optimize in vitro indirect plant regeneration technique considering various culture aspects for callus induction and shoot generation from leaf explants and internodal explants of ashwagandha (Withania somnifera L.).
Leaf explants were collected from Petri - dish grown plants and internodal explants were collected from in vitro grown plants. In the present study callus induction was conducted by using the leaf segments and internodal segments as explants. With proper manipulation of cytokinin and auxin concentrations and combinations in MS medium, it was possible to induce callus from two different explants. It was observed that 2, 4-D was the best auxin for callus induction in ashwagandha. The performance of different concentration of NAA was not as good as 2, 4-D. Among all the treatments of 2, 4-D, maximum 60% callusing rate was found in media having 2.0mg/l 2,4-D from leaf explants.
Calli derived from two types of explants were subcultured in different concentrations and combinations of BA with NAA and KIN with NAA for shoot regeneration. Between the two combinations, KIN + NAA was proved to be more effective than BA + NAA for maximum shoot induction from leaf explants derived calli in ashwagandha.
Among all the treatments of BA with NAA, the media fortified with 3.0 mg/l BA +0.1mg/l NAA were found optimum 40% for leaf explants responded to develop shoots. The maximum number of shoots per callus was also recorded in this media formulation. The highest length of the longest shoot was 2-8 cm in media having 2.0 mg/l BA + 1.0mg/l NAA.
Among all the concentrations and combinations of BA with NAA, the media having 3.0 mg/l BA+1.0 mg/l NAA was found 30% shoot regeneration form internodal segment derived calli. The maximum number of shoots per callus was also recorded in this media composition.
MS medium was supplemented with different concentrations and combinations of KIN with NAA to induce shoot regeneration. The highest 50% leaf segment explants of ashwagandha regenerated shoot in media having 4.0mg/l KIN + 0.5 mg/l NAA. The highest regeneration rate of internodes derived calli were 30% in media having 4.0 mg/l KIN + 0.5mg/l NAA. The maximum number of shoots per callus was also found in this media formulation. In the present investigation 4.0mg/l KIN + 0.5mg/l NAA was recorded as optimum concentration and combination for maximum shoot regeneration and highest number of shoots per callus from both type of explants in ashwagandha. These results indicate that different concentrations and combinations of growth regulators have a great effect on callus induction and plant regeneration of ashwagandha.
CHAPTER VI
6. REFERENCES
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LIST OF TABLES
Table No.
Table 2.2.1 Preparation of stock solutions for growth gegulators.
Table 3.1 Effect of HgCl2 treatment duration for surface sterilization of shoots (containing leaves and internodes) of ashwagandha.
Table 3.2 Effects of different concentrations of 2,4-D in MS medium on callus induction from leaf explants of ashwagandha.
Table 3.3 Effects of different different concentrations of 2, 4-D in MS medium on callus induction from leaf explants of ashwagandha.
Table 3.4 Effects of different concentrations of NAA in MS medium on callus induction from leaf segment explants of ashwagandha.
Table 3.5 Effects of different concentrations of 2, 4-D in MS medium on callus induction from internodal explants of ashwagandha.
Table 3.6 Effects of different concentrations of NAA in MS medium on callus induction from internodal segment explants of ashwagandha.
Table 3.7 Effects of different concentrations and combinations of BA with NAA in MS medium on shoot regeneration from leaf explants derived calli of ashwagandha.
Table 3.8 Effects of different concentrations and combinations of Kin with NAA in MS medium on shoot regeneration from leaf explants derived calli of ashwagandha.
Table 3.9 Effects of different concentrations and combinations of BA with NAA in MS medium on shoot regeneration from internodal segment explants derived calli of ashwagandha.
Table 3.10 Effects of different concentrations and combinations of Kin with NAA in MS medium on shoot regeneration from internodal explants derived calli of ashwagandha.

APPENDIX


APPENDIX-I: List of abbreviations


BA
6-benzyl adenine
0C
Degree celsius
cm
Centimeter
e.g.
exempli gratia, for example
et al.
et alia (and others)
etc.
et cetra, and the rest
F
Degree fahrenheit
Fig./s
Figure(s)
g
Gram
g/l
Gramliter
HCI
Hydrochloric acid
ha
Hector
K cal
Kilo calory
Kg
Kilogram
KIN
Kinetin (6-furfuryl amino purine)
KOH
Potassium Hydroxide
L./Linn.
Linnaeus
Lbs/(inch)2
Pound per square inch
mg
Milligram
mg/l
Milligram/liter
ml
Milliliter
mm
Millimeter
MS
Murashige and Skoog
Min
Minute(s)
NAA
-Naphthalene acetic acid
Na0h
Sodium hydroxide
Na2-EDTA
Sodium salt of ferric ethylene diamine tetra acetate
0.1N
0.1Normal
No.
Number
NPK
Nitrogen, Phosphorus, Potassium
%
Percentage
pH
Negative logarithm of hydrogen ion (H+) concentration
2, 4-D
2, 4-dichlorophenoxy acetic acid
viz.
Videli (Namely)





APPENDIX-II: Formulation of MS medium (Murashige and Skoog, 1962)


Component
Concentration (mg/l)
Major nutrients


KNO3
1900.00

NH4NO3
1650.00

KH2PO4
170.00

CaCl2.2H2O
440.00

MgSO4.7H2O
370.00




Micro nutrients
27.80

FeSO4.7H2O
37.30

Na2-EDTA. 2H2O
22.30

MnSO4.4H2O
6.20

H3BO3
8.60

ZnSO4.7H2O
0.83

Kl
0.25

Na2MoO4.2H2O
0.025

CuSO4.5H2O
0.025




Organic nutrients


Glycine
2.00

Nicotinic acid
0.50

Pyridoxine-HCI
0.50

Thiamine-HCI
0.50

Myoinositol
100.00




Sucrose
30000.00

pH = 5.8